The present invention is of carboxylic cationites with improved sorption capacity, particularly for high molecular weight substances such as proteins, polysaccharides and other macromolecules as well as for improved methods for manufacturing the carboxylic cationites.
Ion exchange is one of the most common procedures for biomolecules isolation and purification, and there are many ion exchanging supports for bio-chromatography. The ion-exchange chromatography technique is based on the ability to separate charged substances by ‘adherence’ to resins carrying the opposite charge. The ion groups of exchange are bound through a covalent bond to the absorbing matrix.
Many currently available background art sorbent materials for ion-exchange chromatography are based on various polysaccharides such as those known under the trade names Sepharose™, Sephadex™ and so forth. Unfortunately these sorbent materials have only limited sorption ability and are not able to fully separate macromolecules, such as proteins, from complex mixtures in the presence of mineral salts. Therefore, such background art sorbent materials are useful only after the stage of removing mineral salts from the mixture, or “desalting”. In addition, the chemical stability and thermostability of these materials is low, and accordingly, they are useful only for a limited number of regeneration cycles.
Carboxylic cationites have a number of advantages over such polysaccharide-based sorbents, and as such are known and widely used for sorption and desorption of various macromolecules such as proteins. One of the advantages of these cationites is that they do not denature the proteins and enzymes during the process of separation. Unfortunately, those carboxylic cationites which are known in the background art largely feature a macroporous structure which has a high flow rate and a low exchange capacity, with limited sorption of macromolecules such as proteins. This structure limits the usefulness of carboxylic cationites for ion exchange chromatography with such macromolecules. The structure of the carboxylic cationites is in turn a result of the method employed for manufacturing these cationites.
U.S. Pat. No. 4,128,032 discloses one such background art method of manufacture for carboxylic cationites. This process involves the co-polymerization of methacrylic or acrylic acids with a suitable crosslinking agent, such as hexahydro-1,3,5-triacryloyltriazine (HTA), N,N′-methylenediacrylamide (MDAA), N,N′-ethylenedimethacrylamide (EDMA), or N,N′-hexamethylenedimethacrylamide (HMDMA) with an initiator of radical polymerization. The resulting reaction mixture is dispersed in high viscosity liquid, either liquid polyethylsiloxane or polymethylsiloxane, as the dispersing medium.
Unfortunately, the carboxylic cationites prepared according to this background art process do not possess sufficient sorption capacity required for the effective separation of macromolecules such as proteins and enzymes. This deficiency may be caused by the phenomena of absorption and surface tension on the phase interface of the reaction mixture and liquid polysiloxane. This tension may decrease the size of pores formed on the outside surface of the granules of carboxylic cationites. Furthermore, such a process for manufacturing the cationites also has the disadvantage of requiring an organic solvent to wash out the dispersion medium, which is a potential safety hazard and which increases the expense of manufacture.
A further disadvantage of this background art method is the migration of components of the reaction mixture (comonomers and acetic acid) into the dispersion medium, which occurs during the stage of copolymerization. Such migration increases the difficulty of preparing carboxylic cationite granules with reproducible structural properties. Thus, the disclosed background art method has a number of disadvantages.
There is thus a need for, and it would be useful to have, a method for manufacturing carboxylic cationites which requires little or no organic solvent in the dispersion medium, and which results in the production of cationites with reproducible structural properties and efficient sorption of macromolecules such as proteins.